This project is directed towards determining a molecular basis for the modulation of G protein-coupled receptor signaling by polyunsaturated phospholipids, in particular those containing 22:6n-3 acyl chains. The membranes of neuronal and retinal tissue are unusually high in phospholipids containing one or two long chain polyunsaturated acyl chains. Docosahexaenoic acid (abbreviated DHA or 22:6n-3) is the major polyunsaturated acyl chain in these tissues. Studies are also focused on the response of these phospholipids to the acute exposure to ethanol. In a study completed this year using rod outer segment disk membrane derived from control and n-3 deficient rats, we have demonstrated differences in the phospholipid acyl chain packing properties that result from the substitution of DPA, 22:5n-6, an n-6 acyl chain for the n-3 DHA acyl chain. These two side chains differ by only a single double bond, yet the substitution was found to have marked impact on acyl chain packing free volume, penetration of the membrane by water molecules, and motion of the fluorescent membrane probe DPH. In addition this substitution produced severe functional consequences for the efficacy of G protein-coupled signaling in the visual transduction pathway. In a second study completed this year we showed that phospholipids derived from trans fatty acids (TFA) have a higher membrane cholesterol partition coefficient than those from cis fatty acids. Incorporation of rhodopsin into TFA-phospholipid bilayers resulted in a higher thermal transition temperature for rhodopsin unfolding and reduced formation of active MII conformation of the receptor upon light activation. Since membrane cholesterol and membrane receptors (e.g. LDL receptor) play essential roles in regulation of cholesterol homeostasis, the combination of higher cholesterol affinity and reduced receptor activation associated with TFA-phospholipids could play an important role in modulation of cholesterol homeostasis that contributes to the elevation of LDL cholesterol. This study is currently under review for publication. We also completed an examination of the effects of membrane phosphatidylethanolamine (PE) concentration on acyl chain order and rhodopsin conformation changes. Rhodopsin was reconstituted in vesicles with varying mol% of di-18:1 PE, with the balance consisting of di-18:1PC. MII production increased with the concentration of di-18:1PE. An optimal level of MII production was detected in membranes containing 30 mol% DOPE. Pure lipid membranes showed increased in acyl chain order as the mol% of DOPE was increased. A novel and surprising finding of this study was that the addition of rhodopsin to the PE-containing bilayers produced more disordered acyl chain packing, and this effect increased with increasing mol% PE. In contrast, the addition of membrane protein to a pure PC bilayer produces more ordered acyl chain packing. This observation indicates that the lipid-protein interactions in mixed bilayers of PC and PE, such as those found in neuronal tissues, are very different from those observed in pure PC bilayers. This study is currently being extended to include biologically significant phospholipids which contain 22:6n-3 acyl chains and both PC and PE headgroups.. The studies carried out here are important in developing an understanding of the role lipid composition in general and 22:6n-3 acyl chains in particular, in domain formation, the modulation of membrane protein function, the heterogeneous distribution of cholesterol in cell membranes, and the health benefits and deficits associated with polyunsaturated fatty acids and trans fatty acids, respectively.